To measure the interaction between immobilized anti-TCR/CD3 Ab and T lymphocytes, characterize the phenomenon in terms of its biochemistry and function, and investigate the role of cytoskeleton in receptor- mediated signalling. Perturbation of the TCR/CD3 with anti-TCR/CD3- antibodies (Ab) is followed by a sequence of biochemical and biological responses similar to those observed subsequent to T-cell interaction with antigen (Ag). Using fluoresceinated Ab, discrete clustering of the TCR/CD3 in defined areas of the cell surface ("patching"), followed by coalescence into a unitary aggregate ("capping") have been observed. Receptor endocytosis usually follows. Ab immobilized onto solid matrices have been used as an alternative to perturbation of the TCR/CD3 with perturbation similar to physiologic conditions, since normal Ag/TCR/CD3 interaction occurs in the context of the cell surface MHC of APC. As opposed to soluble Ag, the rigidity of a solid matrix would effectively prevent receptor mobility and internalization. Consequently, receptor perturbation by immobilized Ab might occur independently at multiple sites of the cell surface. Preventing the process of TCR/CD3 internalization may have biochemical and biological consequences. We have defined conditions and requirements for the interaction of T lymphocytes with immobilized anti-TCR/CD3 Ab. Specific binding of anti-TCR/CD3 Ab-coated beads to 8-5-5 T cells ( CD3+, CD4+, murine T-cell clone) was temperature dependent, augmented by increasing extracellular [Ca2+], and followed a time-course parallel to signal generation, measured as inositol phospholipid (InsPL) hydrolysis. Cytochalasin pretreatment (to disrupt microfilament assembly) inhibited conjugation in a dose-dependent manner, but enhanced InsPL hydrolysis induced by treating 2C11-coated cells with soluble anti-hamster Ab's. Vinca alkaloids, which disrupt microtubuli, were ineffective. Potentiation of InsPL hydrolysis by cytochalasins affected the initial rate and maximal level of the response. Decay rates were unaffected, suggesting that the turn off mechanism is independent of the microskeleton. The effect of cytochalasins did not correlate with the association of the TCR/CD3 complex with detergent-insoluble cytoskeleton, but was associated with a decreased receptor internalization.